In vitro diagnostic assays may be used to measure amounts of an analyte found in a body fluid sample or tissue sample. The analyte must be distinguished from other components found in the sample. Analytes may be distinguished from other sample components by reacting the analyte with a specific receptor for that analyte. Assays that utilize specific receptors to distinguish and quantify analytes are often called specific binding assays.
The most common receptors are antibodies and specific binding proteins such as Intrinsic Factor or Folate Binding Protein. Receptors are characterized by having a reversible specific binding affinity for an analyte or an analogue of that analyte. The analogue generally is an analyte derivative carrying a detectable marker such as an enzyme, fluorescent molecule or other known label but which binds to a receptor with about the same specificity and affinity as the analyte.
In heterogeneous specific binding assays described in the technical and patent literature, the receptor or other assay reagent of the specific binding reaction is often immobilized on a solid phase. Immobilization of these reagents is required to separate the bound components (for example analyte bound to the solid phase through a receptor) from the unbound components.
The various methods by which a receptor or other reagent can be immobilized on a solid phase include adsorption, absorption or covalent bonding. However, many of the solid phase supports used in such assays are not inert, and may sequester proteins and other substances from the sample by non-specific binding. Although glass is a relatively inert substrate, generally it has been found to be unsatisfactory for use in solid phase binding assays. See, for example U.S. Pat. No. 3,790,653 for a discussion of inadequacies of glass substrates.
Recently, however, procedures have been described for immobilizing an essentially soluble immunocomplex of a reagent and an antiserum to the reagent on an inert glass fiber solid phase support. These procedures are disclosed in U.S. Pat. No. 4,517,288, incorporated by reference herein.
In these immunological immobilization procedures, soluble immunocomplexes are prepared by combining at least two immunochemically reactive substances with one another in solution. At least one of such immunochemically reactive materials is selected for its immunochemical specificity for an analyte of interest. For example, if the soluble immunocomplex is to be used in an immunoassay for the detection of TSH, then one component of the immunocomplex is selected for its immunochemical specificity for TSH. A typical example would be an antibody with specificity for TSH, i.e., an anti-TSH antibody. The second component of the immunocomplex could comprise an antibody preparation directed against the anti-TSH antibody. Antiserum to anti-analyte antibodies, for example to mouse anti-TSH antibodies, can be prepared by injecting purified mouse IgG into a host animal (i.e., goat), and thereafter harvesting the antiserum to the mouse IgG. The mouse anti-TSH antibody and the goat antiserum to mouse IgG are thereafter worked up as standard stock solutions.
Once having prepared these stock solutions, a portion of each is combined with the other by addition to a buffered medium. The resulting immunocomplex, in an appropriate volume of buffer, is thereafter spotted onto a delimited area of a glass fiber filter. Alternatively, the two components of the immunocomplex may be applied to the filter as separate buffered solutions and allowed to react in situ. In both instances, the point of application of the immunocomplex defines a reaction zone within the solid phase. The applied immunocomplexes become adsorbed and entrapped within the interstices of the beds of fibers within the glass fiber filter. The method of application can include dispensing of the immunocomplex solution with a manual or automated pipette, or with other automated equipment including assay analyzer instruments. Subsequent to application of the immunocomplex to the solid phase and the elapse of a suitable incubation period, the solid phase is dried under controlled conditions thereby yielding a stable reactive reagent which can be used in any one of a number of solid phase specific binding assay protocols.
Immunological immobilization, although useful in a variety of assay formats, has been noted to include a number of inherent disadvantages. The presence of the additional immunoglobulins on the filter (e.g., antiserum to anti-analyte antibodies) can lead to nonspecific binding of proteinaceous and other biological materials. This can significantly decrease assay sensitivity. Moreover, given the inherent variability of IgG preparations from separate immunizations of the same or different host animals, lot-to-lot variability in titer, purity, specificity and affinity of IgG preparations must be accounted for in manufacturing procedures. Similarly, variability in production of solid phase reagents may be encountered due to the tendency of immunocomplexes to become inhomogeneously distributed within stock solutions. That is, such immunocomplexes, while substantially soluble, may not remain completely soluble and may undergo some settling out of solution over time. Even with periodic mixing of stock solutions, gravitational influences, temperature gradients and other physical influences can cause subtle inhomogeneities within solutions applied to the solid phase reagents.
Starburst dendrimers (manufactured by The Dow Chemical Company) are polymers of spherical or other three-dimensional shapes that have precisely defined compositions and that possess a precisely defined molecular weight. Such dendrimers can be synthesized as water soluble macromolecules through appropriate selection of internal and external moieties. See U.S. Pat. Nos. 4,507,466 and 4,568,737, incorporated by reference herein. Dendrimers may be conjugated with various pharmaceutical materials as well as with various targeting molecules that may function to direct the conjugates to selected body locations for diagnostic or therapeutic applications. See for example, WO 8801178, incorporated by reference herein. Starburst dendrimers have been used to covalently couple synthetic porphyrins (e.g., hemes, chlorophyll) to antibody molecules as a means for increasing the specific activity of radiolabeled antibodies for tumor therapy and diagnosis. Roberts, J. C. et al., Using Starburst dendrimers as Linker Molecules to Radiolabel Antibodies, Bioconjug. Chemistry 1: 305-308 (1990).